Detection of mutation by resolvase cleavage

ABSTRACT

Methods are disclosed for detecting one or more mutations in an isolated test nucleic acid by forming a heteroduplex with a homologous control DNA and contacting the heteroduplex with a resolvase capable of recognizing at least one single base pair mismatch within the heteroduplex. In preferred embodiments of the invention, the resolvase is bacteriophage T4 endonuclease VII.

This is a continuation of application Ser. No. 08/232,530, filed Apr.25, 1994, now abandoned.

BACKGROUND OF THE INVENTION

The ability to detect mutations in coding and non-coding DNA, as well asRNA, is important for the diagnosis of inherited disease. A genemutation can be a single nucleotide change or multiple nucleotidechanges in a DNA sequence encoding an essential protein. A singlenucleotide change or multiple nucleotide changes can result in a frameshift, a stop codon, or a non-conservative amino acid substitution in agene, each of which can independently render the encoded proteininactive. However, a gene mutation can be harmless, resulting in aprotein product with no detectable change in function (i.e. a harmlessgene polymorphism). Mutation in repetitive DNA can also lead to disease,as in, for example, human fragile-X syndrome, spinal and bulbar musculardystrophy, and myotonic dystrophy.

A mutant nucleic acid which includes a single nucleotide change ormultiple nucleotide changes will form one or more base pair mismatchesafter denaturation and subsequent annealing with the corresponding wildtype and complementary nucleic acid. For example, G: A, C: T, C: C, G:G, A: A, T: T, C: A, and G: T represent the eight possible single basepair mismatches which can be found in a nucleic acid heteroduplex,wherein U is substituted for T when a nucleic acid strand is RNA.Nucleic acid loops can form when at least one strand of a heteroduplexincludes a deletion, substitution, insertion, transposition, orinversion of DNA or RNA. Several screening methods have been designed todetect DNA mismatches in a heteroduplex. These methods include RNAse Adigestion, chemical cleavage, as well as PCR- and primer extension-baseddetection methods (reviewed in Cotton, Curr. Opinion in Biotech. 3, 24(1992)).

The resolvases (e.g. X-solvases of yeast and bacteriophage T4, Jensch etal. EMBO J. 8, 4325 (1989)) are nucleolytic enzymes capable ofcatalyzing the resolution of branched DNA intermediates (e.g., DNAcruciforms) which can involve hundreds of nucleotides. In general, theseenzymes are active close to the site of DNA distortion (Bhattacharyya etal., J. Mol. Biol., 221, 1191, (1991)). T4 Endonuclease VII, the productof gene 49 of bacteriophage T4 (Kleff et al., The EMBO J. 7, 1527,(1988)) is a resolvase (West, Annu. Rev. Biochem. 61, 603, (1992)) whichwas first shown to resolve Holliday-structures (Mizuuchi et al., Cell29, 357, (1982)). T4 Endonuclease VII has been shown to recognize DNAcruciforms (Bhattacharyya et al., supra; Mizuuchi et al., supra) and DNAloops (Kleff et al., supra), and it may be involved in patch repair.Bacteriophage T7 Endonuclease I has also been shown to recognize andcleave DNA cruciforms (West, Ann. Rev. Biochem. 61, 603, (1992)).Eukaryotic resolvases, particularly from the yeast Saccharomycescerevisiae, have been shown to recognize and cleave cruciform DNA (West,supra; Jensch, et al., EMBO J. 8, 4325 (1989)).

Other nucleases are known which recognize and cleave DNA mismatches. Forexample, S1 nuclease is capable of recognizing and cleaving DNAmismatches formed when a test DNA and a control DNA are annealed to forma heteroduplex (Shenk et al., Proc. Natl. Acad. Sci. 72, 989, (1975)).However, the rate of cleavage is unacceptably slow (Dodgson et al.,Biochemistry 16, 2374, (1977)). The Mut Y repair protein of E. coli isalso capable of detecting and cleaving DNA mismatches. However, the MutY repair protein is only capable of detecting 50% of the total number ofmutations occurring in a mutant DNA segment (Lu et al., Genomics 14,249, (1992)).

SUMMARY OF THE INVENTION

In general, the invention features a method for detecting one or moremutations in an isolated test nucleic acid which preferentiallyhybridizes to an isolated control DNA. The method includes:

a) denaturing, either independently or together, an isolated testnucleic acid and an isolated control DNA, wherein the denaturing issufficient to form denatured test nucleic acid and denatured controlDNA;

b) annealing the test nucleic acid to the control DNA, wherein theannealing is sufficient to form a heteroduplex between the test nucleicacid and the control DNA;

c) contacting the heteroduplex with a resolvase capable of recognizingat least a single base mismatch in the heteroduplex, the contactingbeing under conditions which permit the resolvase to cause one or moreDNA breaks in the heteroduplex; and

d) detecting the breaks as an indication of the presence of one or moremutations in the isolated test nucleic acid.

In preferred embodiments of this method, the resolvase is abacteriophage resolvase, preferably either bacteriophage T4 EndonucleaseVII or bacteriophage T7 Endonuclease I; or the resolvase is a eukaryoticresolvase, preferably a resolvase from the yeast Saccharomycescerevisiae, more preferably any one of Endo X1, Endo X2 or Endo X3resolvases from the yeast Saccharomyces cerevisiae.

In other preferred embodiments, the test nucleic acid and/or control DNAis derived from any one of a eukaryotic cell, eubacterial cell, abacteriophage, a DNA virus, or an RNA virus. Preferred RNA virusesincludes human T-cell leukemia virus and human immunodeficiency virus,preferably any one of HTLV-I, HTLV-II, HIV-1, or HIV-2. Preferred DNAviruses include any one of the family Adenoviridae, Papovaviridae, orHerpetoviridae.

In other preferred embodiments, the control DNA is isolated from anoncogene or a tumor suppressor gene of a eukaryotic cell, preferably amammalian oncogene or a mammalian tumor suppressor gene. Preferably, themammalian oncogene is any one of the abl, akt, crk, erb-A, erb-B, ets,fes/fps, fgr, fms, fos, jun, kit, mil/raf, mos, myb, myc, H-ras, K-ras,rel, ros, sea, sis, ski, src, or yes oncogenes and the tumor suppressorgene is any one of the p53, retinoblastoma, preferably RB1, adenomatouspolyposis coli, NF-1, NF-2, MLH-1, MTS-1, MSH-2, or human non-polyposisgenes.

In other preferred embodiments, the control DNA is isolated from any oneof the β-globin, phenylalanine hydroxylase, α₁ -antitrypsin,21-hydroxylase, pyruvate dehydrogenase Elα-subunit, dihydropteridinereductase, rhodopsin, β-amyloid, nerve growth factor, superoxidedismutase, Huntington's disease, cystic fibrosis, adenosine deaminase,β-thalassemia, ornithine transcarbamylase, collagen, bcl-2,β-hexosaminidase, topoisomerase II, hypoxanthinephosphoribosyltransferase, phenylalanine 4-monooxygenase, Factor VIII,Factor IX, nucleoside phosphorylase, glucose-6-phosphate dehydrogenase,phosphoribosyltransferase, Duchenne muscular dystrophy, von HippelLindeau, or the mouse mottled Menkes genes of a eukaryotic cell.

In other preferred embodiments, the control DNA is a gene encoding acell cycle control protein, preferably p21, p27, or p16.

In other preferred embodiments, the control DNA is isolated from aeubacterial cell, preferably of the order Spirochaetales, Kinetoplastidaor Actinomycetales, more preferably of the family Treponemataceae,Trypanosomatidae, or Mycobacteriaceae, most preferably of the speciesMycobacterium tuberculosis, Treponema pallidum, Treponema pertenue,Borrelia burgdorferi, or Trypanosoma cruzi.

In other preferred embodiments, the control DNA is a restriction enzymefragment; the control DNA is produced by PCR amplification; the controlDNA is produced by propagation in any one of a eukaryotic cell, abacteriophage, a eubacterial cell, an insect virus, preferably abaculovirus derived vector, or an animal virus. Preferably the animalvirus is a Simian Virus-40 or an adenovirus derived vector.

In a related aspect, the invention features a method wherein prior tostep (c) above, the isolated control DNA and/or isolated test nucleicacid are tagged with at least one detection moiety, the detection moietybeing a radioactive nucleotide, preferably ³² P, ³³ P or an ³⁵ Slabelled nucleotide; biotin; digoxygenin; a luminescent agent,preferably a fluorescent nucleotide, more preferably a fluoresceinlabelled nucleotide; a dye, preferably ethidium bromide, acridineorange, DAPI, a Hoechst dye; or an enzyme. Preferably the control DNA istagged with a detection moiety only at a 5' end.

In another related aspect, the invention features a method whereinbetween step (c) and (d) above, the heteroduplex is tagged with at leastone detection moiety; the detection moiety being a radioactivenucleotide, preferably ³² P, ³³ P or an ³⁵ S labelled nucleotide;biotin; digoxigenin; a luminescent agent, preferably a fluorescentnucleotide, more preferably a fluorescein labelled nucleotide; a dye,preferably ethidium bromide, acridine orange, DAPI, a Hoechst dye; or anenzyme. Preferably, the heteroduplex is tagged with a detection moietyonly at a 5' end.

In still another related aspect, the invention features a method whereinthe heteroduplex is bound at a single 5' end to a solid support,preferably the solid support is an avidin-, or streptavidin-coatedsurface, a streptavidin-ferromagnetic bead, or a nylon membrane.

The invention also features a method for detecting one or more mutationsin an isolated test DNA which preferably hybridizes to an isolatedcontrol DNA. The method includes:

a) denaturing, either independently or together, an isolated test DNAand an isolated control DNA, where the denaturing is sufficient to forma single-stranded test DNA and a single-stranded control DNA;

b) annealing the single-stranded test DNA to the single-stranded controlDNA, the annealing being sufficient to form a heteroduplex between thesingle-stranded test DNA and the single-stranded control DNA;

c) contacting the heteroduplex with bacteriophage T4 Endonuclease VIIunder conditions which permit the endonuclease to recognize one or moreDNA mismatches in the heteroduplex, preferably between one and seven(inclusive) DNA mismatches, and to cause one or more DNA breaks in theheteroduplex; and

d) detecting the DNA breaks as an indication of the presence of one ormore mutations in the isolated test DNA.

In preferred embodiments of this method, the test DNA and/or control DNAis derived from any one of a eukaryotic cell, eubacterial cell, abacteriophage, a DNA virus, or an RNA virus. Preferred RNA virusesinclude human T-cell leukemia virus and human immunodeficiency virus,preferably any one of HTLV-I, HTLV-II, HIV-1, or HIV-2. Preferred DNAviruses include any one of the family Adenoviridae, Papovaviridae, orHerpetoviridae.

In other preferred embodiments, the control DNA is isolated from anoncogene or a tumor suppressor gene of a eukaryotic cell, preferably amammalian oncogene or a mammalian tumor suppressor gene. Preferably, themammalian oncogene is any one of the abl, akt, crk, erb-A, erb-B, ets,fes/fps, fgr, fms, fos, jun, kit, mil/raf, mos, myb, myc, H-ras, K-ras,rel, ros, sea, sis, ski, src, or yes oncogenes and the tumor suppressorgene is any one of the p53, retinoblastoma, preferably RB1, adenomatouspolyposis coli, NF-1, NF-2, MLH-1, MTS-1, MSH-2, or human non-polyposisgenes.

In other preferred embodiments, the control DNA is isolated from any oneof the β-globin, phenylalanine hydroxylase, α₁ -antitrypsin,21-hydroxylase, pyruvate dehydrogenase Elα-subunit, dihydropteridinereductase, rhodopsin, β-amyloid, nerve growth factor, superoxidedismutase, Huntington's disease, cystic fibrosis, adenosine deaminase,β-thalassemia, ornithine transcarbamylase, collagen, bcl-2,β-hexosaminidase, topoisomerase II, hypoxanthinephosphoribosyltransferase, phenylalanine 4-monooxygenase, Factor VIII,Factor IX, nucleoside phosphorylase, glucose-6-phosphate dehydrogenase,phosphoribosyltransferase, Duchenne muscular dystrophy, von HippelLindeau, or the mouse mottled Menkes genes of a eukaryotic cell.

In other preferred embodiments, the control DNA is a gene encoding acell cycle control protein, preferably p21, p27, or p16.

In other preferred embodiments, the control DNA is isolated from aeubacterial cell, preferably of the order Spirochaetales, Kinetoplastidaor Actinomycetales, more preferably of the family Treponemataceae,Trypoanosomatidae, or Mycobacteriaceae, most preferably of the speciesMycobacterium tuberculosis, Treponema pallidum, Treponema pertenue,Borrelia burgdorferi, or Trypanosoma cruzi.

In other preferred embodiments, the control DNA is a restriction enzymefragment; the control DNA is produced by PCR amplification; the controlDNA is produced by propagation in any one of a eukaryotic cell, abacteriophage, a eubacterial cell, an insect virus, preferably abaculovirus derived vector, or an animal virus. Preferably the animalvirus is a Simian Virus-40 or an adenovirus derived vector.

In a related aspect, the invention features a method where prior to step(c) above, the isolated control DNA and/or isolated test DNA are taggedwith at least one detection moiety, the detection moiety being aradioactive nucleotide, preferably ³² P, ³³ P or an ³⁵ S labellednucleotide; biotin; digoxygenin; a luminescent agent, preferably afluorescent nucleotide, more preferably a fluorescein labellednucleotide; a dye, preferably ethidium bromide, acridine orange, DAPI, aHoechst dye; or an enzyme. Preferably the control DNA is tagged with adetection moiety only at the 5' end.

In another related aspect, the invention features a method whereinbetween step (c) and (d) above, the heteroduplex is tagged with at leastone detection moiety; the detection moiety being a radioactivenucleotide, preferably ³² P, ³³ P or an ³⁵ S labelled nucleotide;biotin; digoxigenin; a luminescent agent, preferably a fluorescentnucleotide, more preferably a fluorescein labelled nucleotide; a dye,preferably ethidium bromide, acridine orange, DAPI, a Hoechst dye; or anenzyme. Preferably the heteroduplex is tagged with a detection moietyonly at a 5' end.

Individuals skilled in the art will readily recognize that thecompositions of the present invention can be assembled in a kit for thedetection of mutations. Typically, such kits will include at least oneresolvase capable of detecting a mutation. Preferably, the kit willinclude bacteriophage T4 Endonuclease VII in a suitable buffer and willoptionally include isolated control DNA and pre-formed heteroduplexeswith which to standardize reaction conditions.

The term "isolated nucleic acid," as used herein, refers to a nucleicacid segment or fragment which is not immediately contiguous with (i.e.,covalently linked to) both of the nucleic acids with which it isimmediately contiguous in the naturally occurring genome of the organismfrom which the nucleic acid is derived. The term, therefore, includes,for example, a nucleic acid which is incorporated into a vector. Forexample, DNA can be incorporated into bacteriophage, virus or plasmidvectors capable of autonomous replication. In addition, RNA can beconverted into DNA by reverse transcriptase and subsequentlyincorporated into bacteriophage, virus or plasmid vectors capable ofautonomous replication. The term "isolated nucleic acid" also includes anucleic acid which exists as a separate molecule independent of othernucleic acids such as a nucleic acid fragment produced by chemical meansor restriction endonuclease treatment.

"Homologous," as used herein in reference to nucleic acids, refers tothe nucleotide sequence similarity between two nucleic acids. When afirst nucleotide sequence is identical to a second nucleotide sequence,then the first and second nucleotide sequences are 100% homologous. Thehomology between any two nucleic acids is a direct function of thenumber of matching nucleotides at a given position in the sequence,e.g., if half of the total number of nucleotides in two nucleic acidsare the same then they are 50% homologous. In the present invention, anisolated test nucleic acid and a control nucleic acid are at least 90%homologous. Preferably, an isolated test nucleic acid and a controlnucleic acid are at least 95% homologous, more preferably at least 99%homologous.

A mutation, as used herein, refers to a nucleotide sequence change(i.e., a nucleotide substitution, deletion, or insertion) in an isolatednucleic acid. An isolated nucleic acid which bears a mutation has anucleic acid sequence that is statistically different in sequence from ahomologous nucleic acid isolated from a corresponding wild-typepopulation. Examples of mutation bearing nucleic acid sequences whichstatistically differ in sequence from a homologous or a related nucleicacid isolated from a corresponding wild-type population have beenreported (Balazs, I. et al. Am. J. Hum. Genet. 44: 182 (1989); Sommer,S. S. et al. Mayo Clin. Proc. 64: 1361 (1989); Sommer, S. S. et al.BioTechniques, 12: 82 (1992); Caskey, C. T. et al. Science 256, 784(1992); and references cited therein). The methods of the invention areespecially useful in detecting a mutation in an isolated test or controlnucleic acid which contains between 1 and 50 nucleotide sequence changes(inclusive). Preferably, a mutation in an isolated test or controlnucleic acid will contain between 1 and 10 nucleotide sequence changes(inclusive), more preferably between 1 and 7 nucleotide sequence changes(inclusive).

The term complementary, as used herein, means that two homologousnucleic acids, e.g., DNA or RNA, contain a series of consecutivenucleotides which are capable of forming base pairs to produce a regionof double-strandedness. This region is referred to as a duplex. A duplexmay be either a homoduplex or a heteroduplex that forms between nucleicacids because of the orientation of the nucleotides on the RNA or DNAstrands; certain bases attract and bond to each other to form multipleWatson-Crick base pairs. Thus, adenine in one strand of DNA or RNA,pairs with thymine in an opposing complementary DNA strand, or withuracil in an opposing complementary RNA strand. Guanine in one strand ofDNA or RNA, pairs with cytosine in an opposing complementary strand. Bythe term "heteroduplex" is meant a structure formed between twoannealed, complementary, and homologous nucleic acid strands (e.g. anannealed isolated test and control nucleic acid) in which one or morenucleotides in the first strand is unable to appropriately base pairwith the second opposing, complementary and homologous nucleic acidstrand because of one or more mutations. Examples of different types ofheteroduplexes include those which exhibit a point mutation (i.e.bubble), insertion or deletion mutation (i.e. bulge), each of which hasbeen disclosed in Bhattacharya and Lilley, Nucl. Acids. Res. 17,6821-6840 (1989).

The term "mismatch" means that a nucleotide in one strand of DNA or RNAdoes not or cannot pair through Watson-Crick base pairing and π-stackinginteractions with a nucleotide in an opposing complementary DNA or RNAstrand. Thus, adenine in one strand of DNA or RNA would form a mismatchwith adenine in an opposing complementary DNA or RNA strand. A firstnucleotide cannot pair with a second nucleotide in an opposingcomplementary DNA or RNA strand if the second nucleotide is absent (i.e.an unmatched nucleotide).

A control DNA, as used herein, is DNA having a nucleotide sequence thatis statistically indistinguishable in sequence from homologous DNAobtained from a corresponding wild-type population. A control DNA is atleast 20 nucleotides in length. Preferably, a control DNA is between 100and 40,000 nucleotides in length, more preferably between 150 and 5000nucleotides in length.

A test nucleic acid, as used herein, is DNA or RNA, each of which bearsat least one mutation. A test nucleic acid statistically distinguishablein sequence from homologous DNA obtained from a corresponding wild-typepopulation. A test nucleic acid is at least 20 nucleotides in length.Preferably, a test nucleic acid is between 100 and 40,000 nucleotides inlength, more preferably between 150 and 5000 nucleotides in length.

The formation of a duplex is accomplished by annealing two homologousand complementary nucleic acid strands in a hybridization reaction. Thehybridization reaction can be made to be highly specific by adjustmentof the hybridization conditions (often referred to as hybridizationstringency) under which the hybridization reaction takes place, suchthat hybridization between two nucleic acid strands will not form astable duplex, e.g., a duplex that retains a region ofdouble-strandedness under normal stringency conditions, unless the twonucleic acid strands contain a certain number of nucleotides in specificsequences which are substantially, or completely, complementary. Thus,the phrase "preferentially hybridize" as used herein, refers to anucleic acid strand which anneals to and forms a stable duplex, either ahomoduplex or a heteroduplex, under normal hybridization conditions witha second complementary and homologous nucleic acid strand, and whichdoes not form a stable duplex with other nucleic acid molecules underthe same normal hybridization conditions. "Normal hybridization ornormal stringency conditions" are those hybridization or stringencyconditions which are disclosed below.

Unless defined otherwise, all technical terms and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. All publicationsmentioned herein are incorporated by reference. Examples of thepreferred methods and materials will now be described. These examplesare illustrative only and not intended to be limiting as those skilledin the art will understand that methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents an autoradiograph of CCM and EMC analysis of the PDHElα gene (mutation 13A). This gene includes a C→ A mutation 87 basepairs away from the 5' end of the fragment resulting in detectablemismatches.

FIG. 2 represents an autoradiograph of EMC analysis of the PAH gene(mutation IVS 12NT1) containing a homozygous G→ A mutation 191 basepairs from the 5' end of the PCR fragment resulting in detectablemismatches.

FIG. 3A represents an autoradiograph of CCM and EMC analysis of the PAHgene including a C→G mutation in exon 2. The mutation 73 base pairs fromthe 5' end of the fragment results in detectable mismatches.

FIG. 3B represents an autoradiograph of post-digestion 5'-end labellingof the PAH gene (same fragment as in panel A) including a C→G mutationin exon 2. The mutation results in detectable mismatches.

FIG. 4 represents an autoradiograph of CCM and EMC analysis of the21-hydroxylase A gene (mutation A64) containing a T→A mutation at basepair 1004 of the gene. The mutation 110 base pairs from the 5' end ofthe fragment results in detectable mismatches.

FIG. 5 is an autoradiograph of post-digestion 5'end-labelling of the PSHElα gene (mutation 18A (K387 FS)) containing a homozygous -AA deletion631 base pairs away from the 5' end of the section of the gene studied.The mutation results in detectable mismatches.

FIG. 6 is a schematic drawing illustrating how two heteroduplexes andtwo homoduplexes can arise from the denaturation and annealing of anisolated test nucleic acid (A) and a control DNA (B). The particulartest nucleic acid shown includes one nucleotide mismatch. Thebacteriophage T4 Endonuclease VII recognizes and cleaves single-basepair mismatches in each heteroduplex.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

We have found that bacteriophage T4 endonuclease VII, a resolvase whichis known to cleave Holliday-structures as well as branched or looped DNAstructures, is also capable of recognizing and cleaving heteroduplexeswith a single nucleotide mismatch. This unexpected property ofbacteriophage T4 endonuclease VII has allowed us to rapidly detectmutations in a test nucleic acid which includes at least one mutation.In general, the results indicate that resolvases, in particular,bacteriophage T4 endonuclease VII are useful for detecting mutations ina test nucleic acid, in particular, single base pair mutations.

I. Preparation of Bacteriophage T4 Endonuclease: T4 Endonuclease VII wasprepared as described by Kosak and Kemper (Kosak et al., Eur. J.Biochem. 194, 779, (1990)). Stock solutions were maintained at 3,700U/μl. The T4 Endonuclease VII reaction buffer used in the assay wasprepared as a 10× concentrate as previously described (Kosak et al.,supra). The heteroduplex annealing buffer was prepared as a 2×concentrate as previously described (Cotton, Methods in MolecularBiology 9, 39, (1991)). Conditions for 5'-end labelling DNA withpolynucleotide kinase and general methods for performing denaturingpolyacrylamide gel electrophoresis have been described (Sambrook et al.,Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring HarborLaboratory Press, (1989)).

II. Preparation of Control and Test DNA: Control and test DNA wasprepared by PCR amplification (for a review on PCR see Ausubel, F. M.Current Protocols in Molecular Biology, Chapter 15, Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc (1993)). In theseexperiments, we were interested in detecting mutations in human genomicDNA (e.g. β-globin, phenylalanine hydroxylase (i.e. PAH) and α₁-antitrypsin human genomic DNA), plasmid DNA (e.g. a human21-hydroxylase gene plasmid and a mouse mottled Menkes gene plasmidDNA), or cDNA (e.g. human pyruvate dehydrogenase-Elα subunit (i.e. PDHElα) human dihydropteridine reductase (i.e. DHPR) cDNA, and humanrhodopsin cDNA).

i) PAH gene: The PAH gene mutations IVS12 ntl and R408W were eachindividually PCR amplified using primers A and B as previously described(DiLella et al., Lancet 1, 497, (1988)). Exon 2 of the PAH gene was PCRamplified 5 using the primers 5'-GCA TCT TAT CCT GTA GGAAA-3' (SEQ IDNO: 1) and 5'-AGT ACT GAC CTC AAA TAA GC-3' (SEQ ID NO: 2). The PCRconditions were 105 s at 95° C., 150 s at 58° C. and 3 min at 72° C. for35 cycles.

ii) 21-hydroxylase gene: A 340 bp fragment of the normal (i.e.,wild-type) and mutant 21-hydroxylase B gene were each individuallyamplified using the primers 5'-CTG CTG TGG AAC TGG TGG AA-3' (SEQ ID NO:3) and 5'-ACA GGT AAG TGG CTC AGG TC-3' (SEQ ID NO: 4). A 178 bp sectionof the normal and mutant 21-hydroxylase B gene were each individuallyamplified using the primers 5'-GCT CTT GAG CTA TAA GTG G-3' (SEQ ID NO:5) and 5'-GGG AGG TCG GGC TGC AGC A-3' (SEQ ID NO: 6). The normal21-hydroxylase B gene and the 21-hydroxylase A gene were each amplifiedusing the primers 5'-CTG CAC AGC GGC CTG CTG AA-3' (SEQ ID NO: 7) and5'-CAG TTC AGG ACA AGG AGA GG-3' (SEQ ID NO: 8). PCR conditions foramplification of the 21-hydroxylase A and normal or mutant B genefragments was 105 s at 95° C., 150 s at 62° C. and 3 min at 72° C. TheDNA sequence of the A and B genes, as well as mutant alleles thereof canbe found in Rodriques et al. (EMBO J. 6, 1653-1661 (1987)) and Cotton etal. supra.

iii) Other genes: PCR amplification of the β-globin, α₁ -antitrypsin,and PDH Elα genes have been disclosed. For example, β-globin genemutations (-87 (C→T), frameshift codon 6 (-A), nonsense codon 39 (C→T)and sickle mutation codon 6 (A→T) were each amplified using primers aand b, the IVS II-745 mutation was amplified using primers c and d aspreviously described (Dianzani et al., Genomics 11, 48, (1991)). The α₁-antitrypsin gene was amplified as described (Forrest et al., PrenatalDiagnosis 12, 133, (1992)). The PDH Elα gene (PDH gene mutations 13A,18A and 31A) were amplified using primers PDH-P and PDH-E as previouslydescribed (Dahl et al., Am. J. Hum. Genet. 47, 286, (1990)). The DHPRgene was amplified using the primers GD and F as previously described(Smooker et al., Biochemistry 32, 6443, (1990)). The mouse mottledmenkes gene (MMK) was amplified as described previously. The rhodopsingene (mutation 83 (Asp→Ser in codon 15) was amplified as previouslydescribed (Sullivan et al., Arch. Ophthalmol. 111, 1512, (1993)).

A) Purification of PCR Products: PCR amplification products werepurified by agarose (1.5%) gel electrophoresis, electroeluted ontoWhatman I paper and then eluted with 1× TE (pH 8.0). In all experiments,a control DNA was 5' end-labelled with gamma- ³² P! using T4polynucleotide kinase (Boehringer-Mannheim). Following kinase treatment,the labelled control DNA was ethanol precipitated and the pellet washedthree times with 70% ethanol to remove unincorporated label. The washedDNA pellet was resuspended in distilled water to give approximately 5 ng5' end-labelled DNA/μl.

III. Formation of Control: Test DNA Heteroduplexes:

i) General Method: Single-stranded DNA was prepared by denaturingdouble-stranded test DNA and double-stranded control DNA with heat (i.e.between 90° C. and 100° C. inclusive). Alternatively, denaturation canbe accomplished at lower temperatures by well-known modifications, forexample, by adding glycerol to the denaturation buffer. As is shown inFIG. 6, after renaturation (see below), four double-stranded duplexescan be formed. We have found that bacteriophage T4 endonuclease VII iscapable of cleaving a single base pair mismatch in each heteroduplexshown above, whereas, in general, homoduplexes are not cleaved.Therefore, because each heteroduplex is cleaved by bacteriophage T4endonuclease VII, one or more mismatches in a heteroduplex can bereadily detected. Similarly, unmatched base pairs are also detected in aheteroduplex with this method.

Those skilled in the art will appreciate that denaturation can also beconveniently accomplished by alkali denaturation, followed byrenaturation in neutralizing buffer. Furthermore, when a test nucleicacid is RNA, preferably mRNA, heat denaturation will serve to produceRNA free of intrastrand nucleotide base pairing, thereby rendering thedenatured test RNA a superior template for heteroduplex formation (seeSambrook et al. supra). In another alternative method, control and testnucleic acids can be denatured independently, either by heat or alkali,and mixed together in order to form heteroduplexes.

ii) Heteroduplex Formation: Heteroduplex formation between control andtest DNA was performed in 50 μl (total volume) containing 1× annealingbuffer as previously described (Cotton, supra) except that the annealingtemperature was at 65° C. for 1 hour followed by 20 min at roomtemperature. Calculations of DNA concentration were based on 50-60 ngunlabelled DNA (10X excess) and 5 ng of 5' end-labelled DNA per singlereaction. Heteroduplexes were prepared in `bulk` in 50 μl volume and thepellet resuspended in the appropriate volume of distilled water. Forexample, if six reactions were required then 300 ng mutant DNA and 30 ngof labelled wild type DNA was used. After heteroduplex formation, thepellet was resuspended in 30 μl distilled water (i.e. 5 μl distilledwater is taken per single reaction).

Homoduplexes were prepared in order to determine whether bacteriophageendonuclease VII can non-specifically cleave homoduplexes. An identicalprocedure was performed in order to prepare labelled homoduplex DNAexcept that an excess amount of unlabelled control DNA was annealed withlabelled control DNA.

IV. Enzyme Mismatch Cleavage Assay (i.e. EMC)

5 μl of a labelled homoduplex DNA or a heteroduplex DNA (50-60 ng) waseach added individually to 39 μl of distilled water and 5 μl 10×reaction buffer. Each sample was kept on ice. A cleavage reaction wasinitiated by the addition of 1 μl of the Bacteriophage T4 EndonucleaseVII (100-3,000 U/μl as specified). A stock solution of the endonucleasewas diluted to the required activity in the Bacteriophage T4Endonuclease T4 dilution buffer. After addition of the resolvase, thetubes were spun briefly and incubated at 37° C. for 1 hour unlessotherwise specified. Gel electrophoresis control samples were set up inwhich no bacteriophage T4 Endonuclease was added; the endonuclease beingreplaced with 1 μl of dilution buffer and incubated at 37° C. for 1hour. After incubation, each sample was ethanol precipitated, washed in70% ethanol, dried briefly and thoroughly resuspended by vortexing in 5μl formamide urea loading dye. The 5 μl samples were heated to 100° C.and immediately loaded onto an 8% urea formamide acrylamide sequencinggel. Cleavage products were visualized by autoradiography and the sizesof the products were compared with radiolabelled ΘX174 HaeIII sizemarker. For a discussion on analyzing a nucleic acid sample bydenaturing polyacrylamide gel electrophoresis see Sambrook et al.Molecular Cloning: A Laboratory Approach, Chapter 6 (1989).

V. Post-Digestion 5' End-Labelling of Heteroduplexes:

5' end-labelling of all four original 5' ends of each of the formedduplexes was performed after bacteriophage T4 endonuclease cleavage. Thesensitivity of the EMC technique was increased by labelling these new 5'ends. The post digestion 5' end-labelling method was developed to screenkilobase lengths of DNA in the most time and cost effective manner.

Heteroduplexes were prepared in a 1:1 ratio of unlabelled control andunlabelled test DNA. The conditions used for heteroduplex formation wereidentical to those described above except that 25 ng of each of the DNAsamples was used per reaction. The resolvase digestion was performed on50 ng of unlabelled heteroduplex DNA and the products of digestion were5' end-labelled in a total of 10 μl of (1× kinase buffer), 2 units of 5'polynucleotide kinase and 1 μl of a 1/10 dilution of fresh gamma-³² P!ATP. Following incubation for 45 min at 37° C., the sample was denaturedat 70° C. for 10 min and the reaction mixture was ethanol precipitated.The pellet was washed 3 times in 70% ethanol, dried briefly, resuspendedin 5 μl formamide-urea loading dye, and analyzed on a denaturingpolyacrylamide gel as described above.

For simple and practical use found forming duplexes between equimolarmutant and wild type DNA, cleaving and then 5' end-labelling all 5'termini before polyacrylamide gel electrophoresis a useful modification.Post-digestion 5' end-labelling allows analysis of each strand forendonuclease induced breaks without the need to produce radiolabelledprobes, thus maximizing the opportunity to detect mutations in a testDNA. When using the post-digestion 5' end-labelling method, two cleavedbands were always observed which reflected labelling of the cleavageproducts on both the control and test strands.

Those skilled in the art will appreciate that in each pre- andpost-digestion 5' end-labelling method described above, a homoduplexconsisting of either test DNA or control DNA is digested withbacteriophage T4 endonuclease VII in order to test for non-specificcleavage.

VI. Chemical Cleavage of Mismatch (CCM)

Chemical cleavage of heteroduplexes was used to verify thatheteroduplexes had completely formed. CCM using osmium tetroxide andhydroxylamine was performed as previously described (Cotton, supra).

VII. Detection of DNA Mismatches Using Bacteriophage T4 Endonuclease VII

All four types of single base pair mismatch combinations which formed asa result of heteroduplex formation between different test and controlDNAs were detected in these experiments. DNA mismatch combinations areorganized by type and are summarized in Table 1 below:

                  TABLE 1                                                         ______________________________________                                                             Sets of                                                                       Mismatches                                                         Mutational Change                                                                        Produced                                                 ______________________________________                                        Type 1:     G <-> T or   G.A                                                              A <-> C      T.C                                                  Type 2:     G <-> A or   G.T                                                              T <-> C      A.C                                                  Type 3:     G <-> C      C.C                                                                           G.G                                                  Type 4:     A <-> T      T.T                                                                           A.A                                                  ______________________________________                                    

FIG. 6 schematically describes how a single mutation in a test DNA hasfour chances of being detected in each of the two heteroduplexes formedin these experiments. Each heteroduplex includes two mismatched DNAstrands which can be cleaved by a resolvase, preferably bacteriophage T4Endonuclease VII. To be effective in detecting mutations at least onestrand in the pair of mismatches of each set must be cleaved. In mostcases, excess unlabelled test DNA was used and thus only cleavage of DNAstrands containing the two mismatched bases present in the labelledprobe were assayed. Experimental results are summarized in Table 2below:

                                      TABLE 2                                     __________________________________________________________________________    Summary of the Mutations tested for by EMC as detailed in the text.                                 Total              Non-                                                       Fragment                                                                            Mismatch                                                                            Mismatch                                                                             specific                             Gene       Source.sup.a                                                                       Mutation                                                                            Length (bp)                                                                         Set   Detected                                                                             Cleavage                             __________________________________________________________________________    α.sub.1 -antitrypsin                                                               Genomic                                                                            G → A                                                                        220   G.T/C.A                                                                             C.A    +                                               (Hom)                                                              21-hydroxylase B                                                                         Plasmid                                                                            .sup. T → C.sup.b                                                            340   G.T./C.A     -                                    gene.sup.†                                                                        (Hom)                                                                              A → C                                                                              A.G/T.C                                                                             A.G/T.C                                     (mutation B3 and B4)                                                          21-hydroxylase B gene                                                                    Plasmid                                                                            .sup. T → C.sup.b                                                            178   G.T/C.A                                                                             G.T    -                                    (mutation B3)                                                                            (Hom)                                                              PAH E1α 13A                                                                        cDNA C → A                                                                        797   A.G/T.C                                                                             C.T    -                                               (Hom)                  see FIG. 1                                  PAH (IVS12nt1)                                                                           Genomic                                                                            .sup. G → A.sup.b                                                            254   G.T/C.A                                                                             G.T/C.A                                                                              +                                               (Hom)                  see FIG. 2                                  PAH (IVS12nt1)                                                                           Genomic                                                                            .sup. G → A.sup.b                                                            254   G.T/C.A                                                                             G.T/C.A                                                                              +                                               (Het)                                                              PAH (R408W Exon12)                                                                       Genomic                                                                            .sup. C → T.sup.b                                                            254   G.T/C.A                                                                             G.T/C.A                                                                              +                                               (Het)                                                              PAH (Exon 2)                                                                             Genomic                                                                            C → G                                                                        133   C.C/G.G                                                                             C.C.sup.p see                                                                        +                                               (Het)                  FIGS. 3A, 3B                                β-globin (-87)                                                                      Genomic                                                                            C → G                                                                        627   C.C/G.G                                                                             C.C    -                                               (Hom)                                                              β-globin.sup.†                                                    (IVS H-745)                                                                              Genomic                                                                            C → G                                                                        1377  C.C/G.G                                                                             C.C/G.G                                                                              -                                    (IVS H-16) (Het)                                                                               C → G!                                                                            C.C/G.G                                                                             C.C    -                                    (IVS H-74)       G → T!                                                                            A.G/T.C                                                                             A.G/C.T                                                                              -                                    (IVS H-81)       C → T!                                                                            G.T/C.A                                                                             G.T/C.A                                                                              -                                    (IVS H-666)      T → C!                                                                            G.T/C.A                                                                             G.T/C.A                                                                              -                                    β-globin (codon 39)                                                                 Genomic                                                                            C → T                                                                        627   G.T/C.A                                                                             G.T    -                                               (Hom)                                                              β-globin (Sickle)                                                                   Genomic                                                                            A → T                                                                        627   A.A/T.T                                                                             ND     -                                               (Het)                                                              21-hydroxylase A gene                                                                    Genomic                                                                            T → A                                                                        204   A.A/T.T                                                                             A.A/T.T                                                                              -                                    (mutation A64)                                                                           (Hom)                  See FIG. 4                                  rhodopsin gene (83)                                                                      cDNA A → G                                                                        1300  G.T/C.A                                                                             ?      -                                    Asn 15->Ser                                                                              (Het)                                                              DHPR.sup.c cDNA T → C                                                                        779   G.T/C.A                                                                             G.T    -                                               (Het)                                                              MMK gene.sup.c                                                                           plasmid                                                                             A → G!                                                                      1502  G.T/C.A                                                                             ?      -                                               (X-linked)                                                         MMK gene.sup.c                                                                           plasmid                                                                             C → T!                                                                      1502  C.T/C.A                                                                             ?      -                                               (X-linked)                                                         MMK gene.sup.c                                                                           plasmid                                                                             del 33 bp!                                                                         1502  33 bp loops                                                                         ?      -                                               (X-linked)                                                         PDH E1α18A                                                                         cDNA del AA                                                                              797   TT/AA loops                                                                         TT loopp                                               (Hom)                  See FIG. 5                                  PDH E1α31A                                                                         cDNA del 7 bp                                                                            797   7 bp loops                                                                          ?      -                                               (Hom)                                                              β-globin (Codon 6)                                                                  Genomic                                                                            del A 797   A/T loops                                                                           ND     -                                               (Het)                                                              __________________________________________________________________________     .sup.a Source of control and test DNA was either genomic, plasmid, or cDN     as described in the text. Test samples bear either homozygous (Hom) or        heterozygous (Het) mutations.                                                 .sup.b These mutations are in an identical fragment for their respective      genes.                                                                        .sup.c These mutations were previously unknown.                                 ! These mutations are polymorphisms present in the wildtype population.     ND Mismatch was not detected by EMC.                                          ? The actual mismatch cleaved by EMC could not be determined.                 .sup.† More than one mismatch was detected in the fragment studied     .sup.p Post digestion 5end labelling was performed on these fragments.        - Nonspecific cleavage was not detected in these experiments             

Each mutation presented in Table 2 occurred only once in a test DNAunless otherwise specified. A detailed description of each experiment isdisclosed below. These data describe the results of twelve type 1, threetype 2, four type 3, two type 4 and four deletion mutations tested forcleavage with bacteriophage T4 Endonuclease VII:

i) Type 1 (Mismatch Set G.A/T.C)

The PAH Elα 13A gene includes a C→ A mutation 87 bp 3' to the 5'terminal end of the specific mutant gene studied (i.e. F205L). Using a5' end-labelled control DNA as a probe, the resulting heteroduplexescontained C*.T and A.G* mismatches which could be detected withbacteriophage T4 endonuclease VII (FIG. 1). In FIG. 1, lanes 1, 2 and 3are samples of control homoduplex DNA (i.e. lanes C) after incubationwith hydroxylamine (i.e. lanes HA) for 0, 1 and 2 hrs. Lanes 4, 5 and 6are samples of heteroduplex DNA including test DNA (i.e. lanes 13A)after incubation with hydroxylamine for 0, 1 and 2 hr, respectively.Lanes 7, 8 and 9 are samples of homoduplex DNA (i.e. lanes C) afterincubation with 0, 1000 and 3000 units of T4 Endonuclease VII,respectively. Lanes 10-14 inclusive represent digestion of heteroduplexDNA including test DNA after incubation with 0, 250, 1000, 2000 and 3000units of T4 Endonuclease VII, respectively. In lanes 5 and 6, a 87 bpfragment was observed on a denaturing acrylamide gel due to the factthat hydroxylamine is only capable of modifying a mismatched cytosine.With the EMC (lanes 10-14), a single band slightly larger than 87 bpfragment was observed indicating cleavage 3' of the C* in the C*.Tmismatch.

In figures shown herein, a drawing beneath an autoradiograph is aschematic representation of the two types of heteroduplexes that wereformed in each experiment. A broken line represents a control DNA strandand a straight line represents a test DNA strand. Arrows denoteendonuclease cleavage sites on the end-labelled strand in eachheteroduplex. A (•) represents a radioactively labelled nucleotide. Ashaded triangle (▴) represents the actual cleavage site in a CCMreaction. A superscript (³) refers to band sizes observed by EMC whichwere found to be larger than the expected band sizes determined by CCM.M denotes 5' end-labelled Hae III digested ΘX174 DNA.

ii) Type 2 (Mismatch Set G.T/A.C)

A 254 bp genomic DNA segment from the PAH gene of a patient homozygousfor a G→A mutation (IVS12 (ntl)) was prepared by PCR-amplification. Thisparticular PAH gene mutation occurs 191 bp 3' from the 5' end of the PCRamplified product (see FIG. 2). 5' end-labelled control DNA was annealedto unlabelled test DNA in order to form a heteroduplex. After cleavagewith bacteriophage T4 endonuclease VII, two bands, one slightly largethan 191 bp and another slightly larger than 54 bp band were observed(FIG. 2). In FIG. 2, lanes 1 to 4 inclusive are samples of controlhomoduplex DNA (i.e. lanes C) after incubation with 0, 250, 500 and 1000units of T4 Endonuclease VII and lanes 5 to 8 inclusive are samples ofheteroduplex DNA consisting of control and test DNA after incubationwith 0, 250, 500 and 1000 units of T4 Endonuclease VII, respectively.These autoradiograms indicate DNA mismatches near a G* (G*.T mismatch)and C* (A.C* mismatch) respectively.

iii) Type 3 (Mismatch Set C.C/G.G)

Another mutant PAH gene segment was PCR amplified in order to form testDNA. This PAH mutant gene segment included a heterozygous C→G mutationat base 57 in Exon 2. This particular PAH mutation is 73 bp 3' from the5' end of the 133 bp segment that was analyzed (see FIG. 3A). FIG. 3A isan autoradiograph of CCM and EMC analysis of this test PAH gene segment.Lanes 1, 2 and 3 are samples of control homoduplex DNA (i.e. lanes C)after incubation with hydroxylamine for 0, 1 and 1.5 hr. Lanes 4, 5 and6 are samples of heteroduplex containing control and test DNA (i.e.lanes PAH) after incubation with hydroxylamine for 0, 1 and 1.5 hr,respectively. Lanes 7 to 11 inclusive are samples of control homoduplexafter incubation with 0, 1000, 2000, 2500 and 3000 units of T4Endonuclease VII. Lanes 12 and 13 are samples of PAH heteroduplex afterincubation with 0 and 1000 units of T4 Endonuclease VII, respectively.

FIG. 3B is an autoradiograph of post-digestion 5' end-labelling(described below) of the PAH gene containing the same heterozygous C→Gmutation in Exon 2. Lanes 1, 2 and 3 are samples of control homoduplexDNA (i.e. lanes C) after incubation with 0, 250 and 1000 units of T4Endonuclease VII and lanes 4 to 8 inclusive are samples of heteroduplexcontaining control DNA and test DNA (i.e. PAH) after incubation with 0,100, 250, 500 and 1000 units of T4 Endonuclease VII, respectively.

As can be seen in both FIGS. 3A and 3B, heteroduplex formation between5' end-labelled control DNA and unlabelled test DNA produced C*.C andG.G* mismatches. CCM using hydroxylamine detected only the C*.Ccontaining heteroduplex. For example, the 73 bp 5' end-labelled strandderived from the control DNA probe (in this case the sense strand) wasobserved on denaturing polyacrylamide gel electrophoresis. EMC of thesame heteroduplex showed a related cleavage pattern, except that a bandslightly larger than 73 bp fragment was also observed (FIG. 3A). Thisband results from cleavage near the C*.C heteroduplex.

Heteroduplex cleavage by T4 Endonuclease VII results in new nucleotide3' OH and 5' PO₄ ends. 5'-end labelling involves only labelling the 5'OH ends of amplified DNA with gamma-³² P! ATP using 5' T4 polynucleotidekinase. Hence any fragments 3' to a heteroduplex cleavage site will NOTbe observed on autoradiography. However, post-digestion 5' end-labellingallowed detection of a 60 base pair fragment previously undetected inFIG. 3A. For example, post digestion 5'-end labelling of theheteroduplex analyzed in FIG. 3A, allowed detection of new bandsslightly larger than 60 and 73 bp bands on the autoradiograph (see FIG.3B). Taken together, comparison of FIGS. 3A and 3B showed that by addinga post-digestion 5'-end labelling step after bacteriophage T4endonuclease VII cleavage, an extra cleavage due to the one mutationcould be identified.

iv) Type 4 (Mismatch Set A.A/T.T)

A 204 bp segment of a mutant 21-hydroxylase A gene was PCR amplifiedfrom plasmid DNA (see above). The DNA segment contained a T→ A mutationat nucleotide 1004 of the 21-hydroxylase A gene. This mutation is 110 bp3' from the 5' end of the gene segment. Annealing a single-strandedcontrol DNA to a single-stranded test DNA resulted in a heteroduplexcontaining both A.A* and T*.T mismatches.

EMC with 5' end-labelled control DNA detected two bands on denaturingpolyacrylamide gels; one slightly greater than 110 bp and the otherslightly greater than 94 bp (FIG. 4). In FIG. 4, lanes 1 and 2 representsamples of control homoduplex DNA (i.e. lanes C) after incubation withosmium tetroxide (OT) for 0 and 5 min and lanes 3 and 4 representsamples of control homoduplex DNA after incubation with 0 and 250 unitsof T4 Endonuclease VII. Lanes 5 and 6 represent samples of heteroduplexDNA containing control and test DNA (i.e., 204 bp mutant 21-hydroxylaseA gene segment) after incubation with OT for 0 and 5 minutes. Lanes 7and 8 are samples of after incubation with 0 and 250 units of T4Endonuclease VII, respectively. Lanes 9, 10 and 11 are samples of testDNA after incubation with 500 units of T4 Endonuclease VII for 1, 3 and16 hr. Lanes 12, 13 and 14 are samples of test DNA after incubation with1000 units of T4 Endonuclease VII for 1, 3 and 16 hr at 37°,respectively. These results confirm that bacteriophage T4 endonucleaseVII is capable of recognizing both control DNA strands in aheteroduplex, each of which includes a DNA mismatch. CCM on this sampleshowed only a 110 bp fragment obtained after modification and cleavageof the mismatched T base in the sense strand of the probe.

iv) Type 5: DNA deletions forming a heteroduplex loop

PDH Elα18A is a 797 base pair fragment of a mutant PDH gene whichincludes a deletion of two deoxyadenosines 631 bp away from the 5' endof the gene segment. EMC detected a single band slightly greater than166 bp signifying cleavage near the `TT` loop only (data not shown).Post-digestion 5' end-labelling of the same segment showed two bands,one slightly greater than 166 bp produced by cleavage near the `TT` loopand the other slightly greater than 631 bp produced by cleavage near the`AA` position on the opposing strand (FIG. 5). In FIG. 5, lanes 1, 2 and3 are samples of control homoduplex DNA (i.e. lanes C) after incubationwith 0, 500 and 1000 units of T4 Endonuclease VII. Lanes 4 to 7inclusive are samples of test heteroduplex containing control DNA andtest DNA after incubation with 0, 500, 1000 and 2000 units of T4Endonuclease VII, respectively.

It will be apparent to the art-skilled that the above-described methodsof detecting mutations are not limited to bacteriophage T4 endonucleaseVII detection methods described above. For example, several resolvasesare disclosed below which are known to recognize and cleave DNAcruciforms. The above-described methods may be used to test theseresolvases for the ability to detect mutations in an isolated testnucleic acid. In addition, the art-skilled will recognize that methodsdescribed above are not limited to any specific nucleic acid template,labelling method, or detection technique. Additional embodiments of theinvention are set forth below:

i) Other resolvases: Bacteriophage endonuclease VII is one example of aresolvase known to cleave DNA cruciforms. Additional resolvases withsimilar activity include bacteriophage T7 endonuclease I, and the S.cerevisiae cruciform cleaving enzymes Endo X1, Endo X2, and Endo X3(reviewed in West, S. C. supra). Methods for purifying bacteriophage T7endonuclease I (deMassy, B., et al. J. Mol. Biol. 193: 359 (1987) ),Endo X1 (West, S. C. and Korner, A. PNAS, 82, 6445 (1985); West, S. C.et al. J. Biol. Chem. 262: 12752 (1987)), Endo X2 (Symington, L. S. andKologner, R. PNAS 82: 7247 (1985)), Endo X3 (Jensch F. et al. EMBO J. 8,4325 (1989)) have been disclosed. Preliminary studies have shown thatEndo X3 is capable of detecting C.C mismatches in much the same way asbacteriophage T4 endonuclease VII, indicating that the yeast resolvases,like bacteriophage T4 endonuclease VII, are also capable of recognizingand cleaving near single base-pair mismatches in a heteroduplex. Inorder to examine the properties of bacteriophage T7 endonuclease I andthe yeast resolvases in more detail, each enzyme can be purified bymethods disclosed above. The ability of each purified resolvase todetect mutations may be examined by using the assays described herein.

ii) Additional DNA sequences: The above described methods show thatbacteriophage T4 endonuclease VII is capable of detecting one or morebase-pair mismatches in a heteroduplex. The art-skilled will recognizethat these methods are not limited to any specific nucleic acid sequencedisclosed herein. For example, a DNA restriction fragment of known orunknown DNA sequence which is suspected of bearing at least one DNAmutation, may be used as a test DNA in the formation of a heteroduplex.Preferably the DNA restriction fragment will be at least 20 base pairsin length. More preferably, the DNA restriction fragment will be between50 and 40,000 base pairs in length inclusive, most preferably between100 and 5000 base pairs in length inclusive. In experiments whereparticularly large DNA fragments are analyzed (i.e. larger than 2 kb) itwill be desirable to cleave the fragment with a second restrictionenzyme in order to obtain a fragment of a size suitable for denaturingpolyacrylamide gel electrophoresis (<2 kb). The choice of a secondrestriction enzyme will guided by creating a restriction enzyme map ofthe restriction fragment.

In another example, a test DNA template suspected of harboring at leastone DNA mutation and for which at least a partial DNA sequence is knowncan be used as a source of PCR-amplified test DNA. A DNA template mustinclude: 1) a region suspected of harboring at least one DNA mutationand 2) include sufficient DNA flanking a suspected mutation to serve asa template for DNA oligonucleotide primer hybridization and PCRamplification. The PCR amplified region is the intervening regionbetween the 3' ends of the two DNA oligonucleotide primers hybridized tothe DNA template. This intervening region harbors at least one DNAmutation. As outlined above, PCR amplification is performed by firsthybridizing two DNA oligonucleotide primers to a DNA template harboringat least one DNA mutation, then completing multiple rounds of PCRamplification; the PCR amplified DNA being used as test DNA forheteroduplex formation as described above. The design of the two DNAoligonucleotide primers used to amplify a DNA template and prepare testDNA are guided by the DNA sequence flanking a suspected mutation siteand two important parameters: 1) DNA oligonucleotide primer size, and 2)the size of the intervening region between the 3' ends of the DNAoligonucleotide primers hybridized to a DNA template. Preferably, a DNAoligonucleotide primer will be at least 12 nucleotides in length. Morepreferably, a DNA oligonucleotide primer will be between 15 and 50nucleotides in length inclusive, most preferably between 15 and 25nucleotides in length inclusive. The size of the intervening regionbetween the 3' ends of the two DNA oligonucleotides hybridized to a DNAtemplate will be governed by 1) the well known size limitations oftemplates amplified by PCR, and 2) the resolving power of a particulargel (ie. polyacrylamide or agarose gel) used to detect resolvasecleavage sites (see below). In general, the intervening region betweenthe 3' ends of the two DNA oligonucleotides hybridized to a DNA templatewill be at least 50 base pairs in length inclusive. Recent advances inPCR technology have allowed amplification of up to 40 kb of DNA.Preferably, the intervening region will be between 100 and 40,000 basepairs in length inclusive, more preferably between 150 and 5000 basepairs in length inclusive. Those skilled in the art will appreciate thatwhere the flanking DNA sequence is only partially known, a degenerateDNA oligonucleotide primer may be used to prepare test DNA by PCRamplification.

In another example, template DNA suspected of harboring at least one DNAmutation can be subcloned into a suitable cloning vector and amplifiedusing known DNA oligonucleotide primers which hybridize to the cloningvector and are adjacent to the insertion site of the DNA template. Inthis instance, no template DNA sequence information is required becausethe DNA oligonucleotide primers used for PCR amplification hybridize toa vector of known DNA sequence and not the inserted template DNA. Forexample, the Bluescript™ vector can be used to sub-clone a DNA templateinto an acceptor site according to the manufacturer's instructions(Stratagene Cloning Systems, La Jolla, Calif., Product Catalogue,(1992)). The T7 and T3 DNA primers of the Bluescript vector can be usedto PCR amplify the inserted DNA template (or concomitantly to sequencethe inserted DNA template). Other commercially available sub-cloningvectors may also be used. These include, without limitation, phagelambda based insertion vectors and other prokaryotic and eukaryoticvectors (i.e., bacteriophage, insect virus (baculovirus) or animal virus(SV-40 or adenovirus) based vectors described by Stratagene supra, andSambrook et al. supra). As described above, the PCR amplified test DNAtemplate can be used as a source of test DNA to make heteroduplexes forcleavage with a resolvase, preferably bacteriophage T4 endonuclease VII.In an alternative method, a vector which includes a DNA insert bearingat least one DNA mutation may be first amplified by propagation inbacteria, phage, insect or animal cells prior to PCR amplification (seeSambrook et al. supra). If sufficient DNA is available (i.e. at least 1nanogram), the PCR amplification step can be eliminated.

In yet another example, RNA suspected of bearing at least one mutationmay be purified from cells or tissues by techniques well-known in theart. For example, RNA may be optionally purified by olido-dTchromatography to prepare mRNA (see for example Sambrook et al. supraand Ausubel et al. supra). In cases where ribosomal RNA is the subjectof analysis or a particular mRNA (e.g. collagen) is in abundance,oligo-dT chromatography will not be necessary. Purified RNA or mRNA willbe heat denatured in order to ensure complete single-strandedness andhybridized with control DNA (i.e. a control cDNA) in order to form RNA:DNA heteroduplexes. A method for forming RNA: DNA duplexes are wellknown in the art and have been described in detail (see Sambrook et al.supra, pp. 7.62-7.65). After formation of an RNA: DNA heteroduplex, theEMC method described above can be used to detect mismatches produced bymispairing between the cDNA and the RNA. In preferred embodiments, thecontrol DNA will be 5' end-labelled while the RNA will not be labelled.Alternatively, RNA can be uniformally labelled by adding radiolabelleduracil to living cells or tissues.

The invention is particularly useful for detecting single base pairmutations in cloned DNA, for example, those mutations introduced duringexperimental manipulation of the cloned DNA (e.g. transformation,mutagenesis, PCR amplification, or after prolonged storage or freeze:thaw cycles). As an example, a DNA segment can be used to form aheteroduplex; wherein the control DNA is DNA not subjected toexperimental manipulation and the test DNA is DNA subjected toexperimental manipulation. The methods described herein can be used as aquick and inexpensive screen to check for mutations in any clonednucleic acid.

Those skilled in the art will appreciate that the present invention canalso be used to type bacteria and viruses. By "type" is meant tocharacterize an isogeneic bacterial or viral strain by detecting one ormore nucleic acid mutations that distinguishes the particular strainfrom other strains of the same or related bacteria or virus. Forexample, bacteria or viruses which share specific DNA mutations would beof similar type. As an example, genetic variation of the humanimmunodeficiency virus has led to the isolation of distinct HIV types,each bearing distinguishing gene mutations (Lopez-Galindez et al., PNAS88, 4280 (1991)). Examples of test DNAs of particular interest fortyping include test DNA isolated from viruses of the familyRetroviridae, preferably the human T-lymphocyte viruses or humanimmunodeficiency virus, in particular any one of HTLV-I, HTLV-II, HIV-1,or HIV-2. Those art-skilled will appreciate that retroviral RNA can bereverse transcribed and conveniently subcloned in vaccinia virus vectorsfor the production of DNA (see Yammamoto et al. J. Immunol. 144, 1999;Nixon et al. Nature 33, 484 (1988); Hu, S-L. et al. WO 87/02038; andreferences therein). Other examples of test DNAs of interest for typinginclude DNA viruses of the family Adenoviridae, Papovaviridae, orHerpetoviridae,; as well as test DNA isolated from microorganisms thatare pathogenic to mammals, preferably humans. Preferred microorganismsfor typing include bacteria of the order Spirochaetales, preferably ofthe genus Treponema or Borrelia; the order Kinetoplastida; preferably ofthe species Trypanosoma cruzi; the order Actinomycetales, preferably ofthe family Mycobacteriaceae, more preferably of the speciesMycobacterium tuberculosis; or the genus Streptococcus.

The invention is also useful for detecting mutations in repetitive DNAassociated with a mammalian disease. For example, one or more mutationsin repetitive DNA is associated with human fragile-X syndrome, spinaland bulbar muscular dystrophy, and myotonic dystrophy (Caskey, T. et al.supra). Repetitive DNA from each of these genes can serve as a testnucleic acid in the methods described herein.

iii) Additional labelling Techniques: The above-described methodsdisclose 5' end-labelling of DNA with radioactive phosphorous eitherbefore heteroduplex formation or after heteroduplex formation andcleavage (i.e. post-digestion 5' end-labelling). Those skilled in theart will appreciate that heteroduplexes can be tagged with one or moreradioactive or non-radioactive detection moieties by a variety ofmethods. For example, during PCR amplification of DNA in order to obtainDNA for heteroduplex formation, one or more deoxyribonucleotides (i.e.dNTPs: dA, dG, dC or T) radiolabelled in the α position with radioactivephosphorus or sulfur (e.g. ³² P, ³³ P, or ³⁵ S) can be added to the PCRamplification step in order to internally label test DNA. In general,0.1-50 μCi of one or more radioactively labelled dNTPs can be added to aPCR reaction and excess label removed by Sephadex G-50 columnchromatography (i.e., a spin column). Furthermore, 5' end-labelling oftest DNA either before or after bacteriophage T4 endonuclease VIIdigestion may be accomplished by using radiolabelleddeoxyribonucleotides. Alternatively, test DNA or control DNA can betagged with biotin (Tizard et al. PNAS 87, 4514 (1990)) beforeheteroduplex formation or after heteroduplex formation and cleavage.Methods for the detection of biotinylated DNA after polyacrylamide gelelectrophoresis have been disclosed (Ausubel et al. supra Chapter 7). Inyet another alternative method, test or control DNA may be tagged withfluorescent dNTPs (i.e. fluorescein, see Ansorge, W., et al. Nucl. AcidsRes. 15, 4593 (1987); Johnston-Dow, E., et al. BioTechniques 5, 754(1987); Prober, J., et al. Science 238, 336 (1987)) before heteroduplexformation or after heteroduplex formation and cleavage. In yet anotherexample, the 3'→5' exonuclease activity of certain DNA polymerases, inparticular T4 DNA polymerase, may be used to radiolabel heteroduplex DNAafter cleavage. Cleaved DNA can be analyzed by denaturing polyacrylamidegel electrophoresis and autoradiography.

iv) Additional Detection Techniques:

a) gel techniques: In addition to the well known basic denaturingpolyacrylamide gel electrophoresis technique described above (i.e. 4% to8% polyacrylamide, 7M urea in a 40-cm long gel of uniform thickness seeSambrook et al. supra), a variety of electrophoretic methods areavailable for increasing the resolution of bacteriophage T4 endonucleaseVII cleavage products and in particular, analyzing large cleavageproducts (ie. >2 kb). Denaturing polyacrylamide gels exhibitingincreased resolution have the advantage of allowing a more precisedetermination of the specific site of mutation in a test DNA.Furthermore, such gels allow improved analysis of cleaved DNA. Forexample, wedge-shaped spacers may be used to create a field gradient orincorporate a buffer gradient, an electrolyte gradient, or an acrylamidestep gradient. Formamide may be included in a standard denaturingpolyacrylamide gel or longer gels may also be employed (80 to 100 cm).These electrophoretic techniques have been described in detail (Ausubelet al. supra Chapter 7). Cleavage products larger than 2 kb can bespecifically cut with a restriction enzyme in order to decrease fragmentsize. Those art-skilled will understand that the choice of a particularrestriction enzyme to specifically cut a large cleavage product will begoverned by a restriction enzyme map of the particular DNA to beanalyzed. Alternatively, a large cleavage product can be electrophoresedon a denaturing (i.e. alkaline) agarose gel and directly visualized byreagents (e.g. stains or dyes) which interact with DNA, for examplesilver, ethidium bromide, acridine orange, 4,6-diamidino-2-phenylindol(i.e. DAPI), Hoechst dyes and the like (see Sambrook et al. supra andBassam, B. J. et al. Anal. Biochem. 196, 80 (1991) ), or theelectrophoresed DNA can be transferred to a membrane, for exampleDEAE-cellulose, nylon, or other suitable membrane; the transferred andmembrane-bound DNA being visualized by filter hybridization with aradioactive or non-radioactive (i.e. biotin, digoxigenin, fluorescein)tagged probe, followed by autoradiography, streptavidin-alkalinephosphatase, digoxygenin visualization techniques (see ClonTech ProductCatalogue, 1989/1990; Boehringer Mannheim Catalog (1993)) orfluorescence detection.

ii) Anchoring heteroduplex DNA to a Solid Support: Hemi-biotinylatedtest or control DNA can be prepared by adding one 5' end biotin taggedoligonucleotide primer to a PCR amplification reaction. In such a PCRreaction, only one DNA strand includes a biotin tag at the 5' end. AfterPCR amplification, the test and control DNAs are denatured, eitherindependently or together, annealed, and allowed to form heteroduplexes.The heteroduplexes are subsequently bound to a solid support. Preferredsolid supports include avidin- or streptavidin-coated surfaces, forexample, ovidin- or streptavidin-coated microtitre dishes, orstreptavidin-coated ferromagnetic beads. Methods for anchoringhemibiotinylated DNA to magnetic beads have been described (Hultman, T.et al. BioTechniques 10, 84 (1991); Kaneoka, H. et al. BioTechniques 10,30 (1991)). After obtaining a biotinylated heteroduplex anchored to amagnetic bead, the anchored heteroduplex is cleaved on both strands witha resolvase, preferably bacteriophage T4 endonuclease VII. Aftercleavage, newly exposed 5' ends are tagged with one or more detectionmoieties as described above, preferably biotin is used to tag newlyexposed 5' ends, and the magnetic beads are concentrated by microfugecentrifugation. The supernatant or beads can be assayed for biotinylated(i.e. cleaved) DNA by gel electrophoresis (see above), or moreconveniently by the use of streptavidin-alkaline phosphatasevisualization techniques (Bethesda Research Laboratories Catalogue andReference Guide, (1989); Clontech Catalog (1989/1990)).

In an alternative method, hemi-biotinylated DNA tagged at one 5' endwith biotin can be tagged with an additional detection moiety at theother 5' end after binding to an avidin- or streptovidin-coated surface,preferably a fluorescent nucleotide (i.e. a fluorescein labellednucleotide, see Clontech Catalog (1989/1990) is tagged to the other 5'end or a radioactive nucleotide is used to tag the other 5' end. Afterbinding to a surface which includes avidin or streptavidin, followed byresolvase cleavage, preferably cleavage with bacteriophage T4endonuclease VII, the supernatant can be analyzed for fluorescence orradioactivity.

In another alternative method, a DNA oligonucleotide primer for PCR canbe tagged with a detection moiety such as digoxigenin. Afteramplification and duplex formation, tagged DNA is anchored to a surfacebearing an antibody capable of binding digoxigenin (see BoehringerMannhelm Catalog (1993)). In yet another alternative method, DNA can betagged with a detection moiety such as an oligo dT tail, and bound to anylon support via ultraviolet light crosslinking of the oligo-dT tail tothe nylon support. The unique nucleic acid sequence is held to the nylonsupport through the oligo-dT tail and is available for duplex formation.In each of these two methods, DNA can be further tagged at an unboundend with biotin, a fluorescent nucleotide, a radioactive nucleotide,then, after duplex formation, cleaved with a resolvase, preferablybacteriophage T4 endonuclease VII. After cleavage, released nucleic acidcan be detected by appropriate methods described above.

Those skilled in the art will appreciate that a variety of methods existfor adding a detection moiety to DNA. For example, Clontech hasdisclosed amino modifiers, and thiol modifiers which could be used totag DNA with biotin, FITC or other fluorophores (Clontech Catalog(1989/1990)).

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      GCATCTTATCCTGTAGGAAA20                                                        (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      AGTACTGACCTCAAATAAGC20                                                        (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      CTGCTGTGGAACTGGTGGAA20                                                        (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      ACAGGTAAGTGGCTCAGGTC20                                                        (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      GCTCTTGAGCTATAAGTGG19                                                         (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      GGGAGGTCGGGCTGCAGCA19                                                         (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      CTGCACAGCGGCCTGCTGAA20                                                        (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      CAGTTCAGGACAAGGAGAGG20                                                        __________________________________________________________________________

What is claimed is:
 1. A method for detecting in an isolated testnucleic acid one or more mutations diagnostic of a disease or condition,said isolated test nucleic acid being derived from a eukaryotic cell,and preferentially hybridizing to an isolated control nucleic acid, saidmethod comprising:a) isolating or providing by denaturation asingle-stranded test nucleic acid and a single-stranded control nucleicacid; b) annealing said single-stranded test nucleic acid to saidsingle-stranded control nucleic acid, wherein said annealing issufficient to form a duplex between said single-stranded test nucleicacid and said single-stranded control nucleic acid; c) contacting saidduplex with a resolvase, wherein said resolvase is capable ofrecognizing and cleaving all eight types of mismatches and wherein saidcontacting is under conditions which permit said resolvase to cause oneor more breaks in said duplex; and d) detecting said one or more breaksas an indication of the presence in said test nucleic acid of one ormore mutations which are diagnostic of said disease or condition.
 2. Amethod for detecting one or more mutations in an isolated test nucleicacid which is derived from a eukaryotic cell, a eubacterial cell, abacterial cell, a mycobacterial cell, a bacteriophage, a DNA virus, oran RNA virus, and which preferentially hybridizes to an isolated controlnucleic acid, said method comprising:a) generating by amplification saidisolated test nucleic acid; b) denaturing said amplified test nucleicacid to provide a single-stranded test nucleic acid; c) isolating orproviding by denaturation a single-stranded control nucleic acid; d)annealing said single-stranded test nucleic acid to said single-strandedcontrol nucleic acid, wherein said annealing is sufficient to form aduplex between said single-stranded test nucleic acid and saidsingle-stranded control nucleic acid; e) contacting said duplex with aresolvase, wherein said resolvase is capable of recognizing and cleavingall eight types of mismatches and wherein said contacting is underconditions which permit said resolvase to cause one or more breaks insaid duplex; and f) detecting said one or more breaks as an indicationof the presence in said test nucleic acid of one or more mutations insaid test nucleic acid.
 3. The method of claim 1 or 2, wherein saidresolvase is a bacteriophage or a eukaryotic resolvase.
 4. The method ofclaim 3, wherein said bacteriophage resolvase is either bacteriophage T4Endonuclease VII or bacteriophage T7 Endonuclease I.
 5. The method ofclaim 3, wherein said eukaryotic resolvase is isolated fromSaccharomyces cerevisiae.
 6. The method of claim 5, wherein saidSaccharomyces cerevisiae resolvase is any one of Endo X1, Endo X2 orEndo X3.
 7. The method of claim 1 or 2, wherein said control nucleicacid or DNA is isolated from any one of a eukaryotic cell, a eubacterialcell, a bacterial cell, a mycobacterial cell, a bacteriophage, a DNAvirus, or an RNA virus.
 8. The method of claim 7, wherein said RNA virusis a human T-cell leukemia virus or a human immunodeficiency virus. 9.The method of 7, wherein said DNA virus is from any one of the familyAdenoviridae, Papovaviridae, or Herpetoviridae.
 10. The method of claim7, wherein said test or said control nucleic acid or DNA is isolatedfrom any one of the β-globin, phenylalanine hydroxylase, α₁-antitrypsin, 21-hydroxylase, pyruvate dehydrogenase Elα-subunit,dihydropteridine reductase, rhodopsin, β-amyloid, nerve growth factor,superoxide dismutase, retinoblastoma, Huntington's disease, cysticfibrosis, adenosine deaminase, β-thalassemia, ornithinetranscarbamylase, collagen, bcl-2, β-hexosaminidase, topoisomerase II,hypoxanthine phosphoribosyltransferase, phenylalanine 4-monooxygenase,Factor VIII, Factor IX, nucleoside phosphorylase, glucose-6-phosphatedehydrogenase, phosphoribosyltransferase, Duchenne muscular dystrophy,von Hippel Lindeau, or the mouse mottled Menkes gene of a eukaryoticcell.
 11. The method of claim 7, wherein said test or said controlnucleic acid or DNA is isolated from an oncogene or a tumor suppressorgene of a eukaryotic cell.
 12. The method of claim 11, wherein saidoncogene is any one of the abl, akt, crk, erb-A, erb-B, ets, fes/fps,fgr, fms, fos, jun, kit, mil/raf, mos, myb, myc, H-ras, K-ras, rel, ros,sea, sis, ski, src, or yes oncogenes.
 13. The method of claim 11,wherein said tumor suppressor gene is any one of the p53,retinoblastoma, adenomatous polyposis coli, NF-1, NF-2, humannon-polyposis coli, MLH-1, MTS1, or MSH-2 genes.
 14. The method of claim7, wherein said eubacterial cell is from any one of the orderSpirochaetales, Kinetoplastida or Actinomycetales.
 15. The method ofclaim 7, wherein said eubacterial cell is from any one of the familyTreponemataceae, Trypanosomatidae, or Mycobacteriaceae.
 16. The methodof claim 15, wherein said eubacterial cell is from any one of thespecies Mycobacterium tuberculosis, Borrelia burgdorferi, Treponemapallidum, or Trypanosoma cruzi.
 17. The method of claim 1 or 2, whereinprior to said contact with said resolvase, said control nucleic acid istagged with at least one detection moiety.
 18. The method of claim 17,wherein said detection moiety is any one of a radioactive nucleotide,biotin, digoxygenin, a luminescent agent, a dye, or an enzyme.
 19. Themethod of claim 18, wherein said control DNA is tagged only at a 5' end.20. The method of claim 1 or 2, wherein between said contact with saidresolvase and said detection step, said duplex is post-digestion 5'end-labeled with at least one detection moiety.
 21. The method of claim20, wherein said heteroduplex comprises at least one of a radioactivenucleotide, biotin, a luminescent agent, or an enzyme.
 22. The method ofclaim 1 or 2, wherein said test or said control nucleic acid or DNA is arestriction enzyme fragment.
 23. The method of claim 1 or 2, whereinsaid test or said control nucleic acid or DNA is produced by PCRamplification.
 24. The method of claim 1 or 2, wherein said test or saidcontrol nucleic acid or said DNA is produced by propagation in aeukaryotic cell, a eubacterial cell, a bacterial cell, or a phage. 25.The method of claim 1 or 2, wherein said duplex is bound to a solidsupport at one 5' end.
 26. The method of claim 7, wherein said controlDNA is isolated from a gene encoding a cell cycle control protein.
 27. Amethod for detecting one or more mutations in an isolated test nucleicacid which is at least 100 nucleotides in length and whichpreferentially hybridizes to an isolated control nucleic acid, saidmethod comprising:a) generating by amplification said isolated testnucleic acid; b) denaturing said amplified test nucleic acid to providea single-stranded test nucleic acid; c) isolating or providing bydenaturation a single-stranded control nucleic acid; d) annealing saidsingle-stranded test nucleic acid to said single-stranded controlnucleic acid, wherein said annealing is sufficient to form a duplexbetween said single-stranded test nucleic acid and said single-strandedcontrol nucleic acid; e) contacting said duplex with a resolvase,wherein said resolvase is capable of recognizing and cleaving all eighttypes of mismatches and wherein said contacting is under conditionswhich permit said resolvase to cause one or more breaks in said duplex;and f) detecting said one or more breaks as an indication of thepresence of one or more mutations in said test nucleic acid.
 28. Amethod for detecting in an isolated test nucleic acid one or moremutations diagnostic of a disease or condition, said isolated testnucleic acid being at least 100 nucleotides in length and preferentiallyhybridizing to an isolated control nucleic acid, said methodcomprising:a) isolating or providing by denaturation a single-strandedtest nucleic acid and a single-stranded control nucleic acid; b)annealing said single-stranded test nucleic acid to said single-strandedcontrol nucleic acid, wherein said annealing is sufficient to form aduplex between said single-stranded test nucleic acid and saidsingle-stranded control nucleic acid; c) contacting said duplex with aresolvase, wherein said resolvase is capable of recognizing and cleavingall eight types of mismatches and wherein said contacting is underconditions which permit said resolvase to cause one or more breaks insaid duplex; and d) detecting said one or more breaks as an indicationof the presence in said test nucleic acid of one or more mutations whichare diagnostic of said disease or condition.
 29. The method of claim 28or 27, wherein said test or said control nucleic acid is isolated fromany one of a eukaryotic cell, a eubacterial cell, a bacterial cell, amycobacterial cell, a bacteriophage, a DNA virus, or an RNA virus. 30.The method of claim 28 or 27, wherein said resolvase is a bacteriophageor a eukaryotic resolvase.
 31. The method of claim 30, wherein saidbacteriophage resolvase is bacteriophage T4 Endonuclease VII.
 32. Themethod of claim 28 or 27, wherein said test nucleic acid is between 150and 5000 nucleotides in length.
 33. The method of claim 29, wherein saidRNA virus is a human T-cell leukemia virus or a human immunodeficiencyvirus.
 34. The method of claim 29, wherein said DNA virus is from anyone of the family Adenoviridae, Papovaviridae, or Herpetoviridae. 35.The method of claim 29, wherein said test or said control nucleic acidis isolated from any one of the β-globin, phenylalanine hydroxylase, α₁-antitrypsin, 21-hydroxylase, pyruvate dehydrogenase Elα-subunit,dihydropteridine reductase, rhodopsin, β-amyloid, nerve growth factor,superoxide dismutase, retinoblastoma, Huntington's disease, cysticfibrosis, adenosine deaminase, β-thalassemia, ornithinetranscarbamylase, collagen, bcl-2, β-hexosaminidase, topoisomerase II,hypoxanthine phosphoribosyltransferase, phenylalanine 4-monooxygenase,Factor VIII, Factor IX, nucleoside phosphorylase, glucose-6-phosphatedehydrogenase, phosphoribosyltransferase, Duchenne muscular dystrophy,von Hippel Lindeau, or the mouse mottled Menkes gene of a eukaryoticcell.
 36. The method of claim 29, wherein said test or said controlnucleic acid is isolated from an oncogene, a tumor suppressor gene, or acell cycle control gene of a eukaryotic cell.
 37. The method of claim36, wherein said oncogene is any one of the abl, akt, crk, erb-A, erb-B,ets, fes/fps, fgr, fms, fos, jun, kit, mil/raf, mos, myb, myc, H-ras,K-ras, tel, ros, sea, sis, ski, src, or yes oncogenes.
 38. The method ofclaim 36, wherein said tumor suppressor gene is any one of the p53,retinoblastoma, adenomatous polyposis coli, NF-1, NF-2, humannon-polyposis coli, MLH-1, MTS1, or MSH-2 genes.
 39. The method of claim36, where said cell cycle control protein is p21, p27, or p16.
 40. Themethod of claim 29, wherein said eubacterial cell is from any one of theorder Spirochaetales, Kinetoplastida, or Actinomycetales.
 41. The methodof claim 29, wherein said eubacterial cell is from any one of the familyTreponemataceae, Trypanosomatidae, or Mycobacteriaceae.
 42. The methodof claim 41, wherein said eubacterial cell is from any one of thespecies Mycobacterium tuberculosis, Borrelia burgdorferi, Treponemapallidum, or Trypanosoma cruzi.
 43. The method of claim 28 or 27;wherein prior to said contact with said resolvase, said control nucleicacid is tagged with at least one detection moiety.
 44. The method ofclaim 43, wherein said detection moiety is any one of a radioactivenucleotide, biotin, digoxygenin, a luminescent agent, a dye, or anenzyme.
 45. The method of claim 43, wherein said control nucleic acid istagged only at a 5' end.
 46. The method of claim 28 or 27, whereinbetween said contact with said resolvase and said detection step, saidduplex is post-digestion 5' end-labeled with at least one detectionmoiety.
 47. The method of claim 46, wherein said duplex comprises atleast one of a radioactive nucleotide, biotin, a luminescent agent, afluorescent agent, or an enzyme.
 48. The method of claim 28 or 27,wherein said control nucleic acid is a restriction enzyme fragment. 49.The method of claim 28 or 27, wherein said control nucleic acid isproduced by PCR amplification.
 50. The method of claim 1 or 2, whereinsaid resolvase is bacteriophage T4 Endonuclease VII.
 51. The method ofclaim 26, wherein said cell cycle control protein is p21, p27, or p16.52. A method for typing a pathogenic microorganism strain, said methodcomprising:a) isolating or providing by denaturation a single-strandedtest nucleic acid derived from said pathogenic microorganism strain; b)annealing said single-stranded test nucleic acid to a secondsingle-stranded nucleic acid having a known pathogenic microorganismsequence, wherein said annealing is sufficient to form a duplex betweensaid single-stranded nucleic acids; c) contacting said duplex with aresolvase, wherein said resolvase is capable of recognizing and cleavingall eight types of mismatches and wherein said contacting is underconditions which permit said resolvase to cause one or more breaks insaid duplex; and d) detecting said one or more breaks as an indicationof the presence in said test nucleic acid of one or more mismatches,said mismatches indicating a nucleic acid sequence in said pathogenicmicroorganism which differs from said known pathogenic microorganismsequence.
 53. The method of claim 52, wherein said pathogenicmicroorganism is a bacterium.
 54. The method of claim 52, wherein saidpathogenic microorganism is a virus.
 55. The method of claim 54, whereinsaid virus is a retrovirus.
 56. The method of claim 55, wherein saidvirus is a human immunodeficiency virus.
 57. The method of claim 55,wherein said virus is HIV-1, HIV-2, HTLV-I, or HTLV-II.
 58. The methodof claim 52, wherein said pathogenic microorganism is a DNA virus. 59.The method of claim 52, wherein said pathogenic microorganism is chosenfrom any one of the family Adenoviridae, Papovaviridae, Herpetoviridae,or Mycobacteriaceae.
 60. The method of claim 52, wherein said pathogenicmicroorganism is chosen from any one of the order Spirochaetales,Kinetolplastida, or Actinomycetales.
 61. The method of claim 52, whereinsaid pathogenic microorganism is chosen from any one of the genusTreponema, Borrelia, or Streptococcus.
 62. The method of claim 52,wherein said pathogenic microorganism is chosen from any one ofTrepanosoma cruzi or Mycobacterium tuberculosis.
 63. The method of claim52, wherein said pathogenic microorganism is pathogenic to a mammal. 64.The method of claim 63, wherein said mammal is a human.
 65. The methodof claim 52, wherein said resolvase is a bacteriophage or a eukaryoticresolvase.
 66. The method of claim 65, wherein said bacteriophageresolvase is bacteriophage T4 Endonuclease VII.
 67. The method of claim52, wherein said test nucleic acid is between 150 and 5000 nucleotidesin length.
 68. The method of claim 52, wherein prior to said contactwith said resolvase, said control nucleic acid is tagged with at leastone detection moiety.
 69. The method of claim 52, wherein said detectionmoiety is any one of a radioactive nucleotide, biotin, digoxygenin, aluminescent agent, a dye, or an enzyme.
 70. The method of claim 52,wherein said control nucleic acid is tagged only at a 5' end.
 71. Themethod of claim 52, wherein between said contact with said resolvase andsaid detection step, said duplex is post-digestion 5' end-labeled withat least one detection moiety.
 72. The method of claim 52, wherein saidduplex comprises at least one of a radioactive nucleotide, biofin, aluminescent agent, a fluorescent agent, or an enzyme.
 73. The method ofclaim 52, wherein said control nucleic acid is a restriction enzymefragment.
 74. The method of claim 52, wherein said test nucleic acid orsaid control nucleic acid is produced by PCR amplification.